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L2-Transcription.Pdf Transcription BIOL201 Daniel Gautheret [email protected] • Chapitre 1: Transcription et RNA polymérase • Chapitre 2: Initiation, élongation et terminaison • Chapitre 3: Régulation: l’exemple procaryote • Chapitre 4: Maturation de l’ARN chez les eucaryotes V. 2012.0 Chapitre 1 Transcription et RNA polymérase 2 L’intuition de la transcription Chez les eucaryotes: ADN dans le noyau Machinerie de synthèse dans le cytoplasme Hypothèse d’un l’intermédiaire ARN 3 Etapes-clé de la découverte de l’ARN messager • 1957: concept d’un « adaptateur ARN » (Crick) • 1956-57: Découverte progressive d’une nouvelle fraction d’ARN, polymorphe • 1959-60: découverte d’une « ARN polymérase » capable de synthétiser de l’ARN à partir d’ADN. • 1961. Mise au point des techniques d’hybridation ADN- ARN sur filtre de nitrocellulose. -> l’ARN est complémentaire d’un seul brin d’ADN • 1961. Démonstration de l’existence d’un ARN messager (Jacob & Monod) • 1961. Purification d’ARN polymérase bactérienne et synthèse in vitro d’ARN à partir de matrice ADN 4 Rappel: différences ADN/ARN ARN ADN ribose désoxyribose 5 Bases de l’ADN et de l’ARN Base puriques ADN: T ARN: U Base pyrimidiques 6 Structure des ARN • Molécule ordonnée linéaire O • Orientée 5’P->3’OH P • Grand nombre de O H2C structures secondaires possibles 7 Décodage du gène dans la cellule bactérienne ADN ARN polymérase Ribosome grande sous-Unité ribosome ARNm Ribosome petite sous-Unité Protéine ARNr ARNt aminoacides Membrane cellulaire 8 Les principaux ARN dans une bactérie • ARN ribosomiques (ARNr) – Grande sous-unité 23S (3000nt) et 5S (120nt) – Petite sous-unité 16S (1500nt) – Composition en bases: 25%A 21%U 32%G 22%C • ARN de transfert (ARNt) – 4S (70nt) • ARN messager (ARNm) – Instable (demi-vie courte) – Grande variété de molécules – Entre 300 et 5000 nt – Taux de synthèse élevé 9 Décodage du gène dans la cellule eucaryote ADN ARN polymérase ARNr Pré-ARNm ARNt Maturation ARNm ribosome Protéine Membrane cellulaire 10 Distribution des ARN dans une cellule eucaryote Cytoplasme Noyau • ARN ribosomiques (ARNr) • Distribution différente du – Grande sous-unité 28S et 5,8S cytoplasme – Petite sous-unité 18S • ARN « géants » (10000 à 100000nt) • ARN de transfert (ARNt) • Turn-over rapide – 4S • ARN messager (ARNm) – Plus stables qu’ARNm bactériens – Grande variété de molécules – Entre 300 et 5000 nt Idée d’ARN précurseurs présents dans le noyau 11 ARN présents dans une cellule typique de mammifère (fibroblaste de souris en culture) ARN cellulaire ARN total (%) à Synthèse l’équilibre totale (%) Précurseurs nucléaires d’ARNr 4 39 ARNr cytoplasmique 71 - Pré-ARNm 7 58 ARNm cytoplasmique 3 ! - Petits ARN stables (surtout ARNt) 15 3 Toute la variété des ARNm est comprise dans ces 3% ! 12 Comparaison transcription/réplication: copie des 2 brins d’ADN au cours de la réplication From Wikipedia: DNA replication 13 Comme pour la réplication: polymérase synthétise de 5’ vers 3’ 5’ ARN synthétisé Matrice ADN 3’ 14 Copie d’un seul brin d’ADN par l’ARN polymérase 5’ T A G T A C G T C A A T A C T G G C C G T G T G A 3’ A T C A T G C A G T T A T G A C C G G C A C A C T 3’ 5’ T A A C A T G 5’ 5’ C T G 3’ T A G T A AC C G C C G T G T G A G U A T C A T G C C G G C A C A C T 3’ A G C 5’ A G U C T A Brin matrice = C A T U A brin transcrit G A T bulle 15 Les gènes peuvent résider sur un brin ou l’autre de l’ADN • Exemple de transcription dans une région génomique: il y a des gènes sur les deux brins. Sens de la transcription sur le brin matrice ADN 5’ ATG 3’ 3’ 5’ AUG Sens de la transcription sur le brin matrice 16 Les étapes de la transcription Site d’initiation Site de terminaison Initiation Elongation Terminaison 17 Structure de l’ARN polymerase de T7 18 Structure de l’ARN polymerase bactérienne bulle 19 Structure de l’ARN polymérase d’E. coli β’ β’ α α α + σ α σ ω β ω β Core enzyme Holoenzyme 7000 molécules/cellule alpha rpoA 36500 Coeur (Core) beta rpoB 151000 site catalytique beta’ rpoC 155000 liaison omega rpoZ 11000 assemblage du core Complet (Holo) sigma rpoD 70000 interaction avec promoteur 20 Les 3 ARN polymérases eucaryotes 21 Activités des 3 ARN polymérases eucaryotes • Pol I: la plus active. Synthétise les ARNr sauf 5S. Dans le nucléole. • Pol II. Synthétise les précurseurs d’ARNm. Dans le noyau. • Pol III. Activité faible. Synthétise les ARNt et autres petits ARN nucléaires (snRNA), et ARNr 5S. Dans le noyau 22 Les 3 ARN polymérases de Saccharomyces cerevisiae β ’−−> β −−> α −−> ω −−> 23 24 Pour la structure de la RNA polymérase II de levure en cours de transcription 25 Saccharomyces cerevisiae RNA POLYMERASE II de 26 27 Structure de l’ARN polymérase II : Longueur considérable du domaine C-terminal (queue CTD) CTD: répétition d’une cinquantaine de séquences “Tyr-Ser-Pro-Thr-Ser-Pro-Ser” 28 Initiation Début de transcription ARN polymérase • La polymérase se fixe au promoteur sur l’ADN duplex promoteur • La polymérase dénature le duplex. Formation de la bulle de transcription • La polymérase catalyse la liaison phosphodiester des deux premiers rNTP 29 Elongation Zone hybride ADN/ARN • La polymérase avance 3’>5’ sur le brin matrice en dénaturant le duplex et en ajoutant des rNTP à l’ARN 5’ ARN natif Terminaison Fin de transcription • Au site d’arrêt de transcription, la polymérase relargue l’ARN terminé et se dissocie de l’ADN ARN terminé 30 Chapitre 2: Initiation, Elongation et Terminaison de la transcription 31 Initiation Début de transcription ARN polymérase • La polymérase se fixe au promoteur sur l’ADN duplex promoteur • La polymérase dénature le duplex. Formation de la bulle de transcription • La polymérase catalyse la liaison phosphodiester des deux premiers rNTP 32 Initiation chez les bactéries 33 Structure d’un promoteur bactérien Promoteur -35 -10 Région transcrite Séquence TTGACA N TATAAT N5-9 A/G consensus 17 αN β’ αC αN β αC σ -35 -10 Reconnaissance du promoteur par le facteur sigma 34 Caractérisation de la région promotrice (Modification des bases, Mutations des paires de bases) Modification empêchant Start la liaison de -35 -10 la RNA pol Modification évitée en présence de RNA pol Mutation nuisant à l’activité du promoteur Points de contact déduits La plupart des points de contacts se trouvent sur la même face de l’ADN 35 Reconnaissance d’un promoteur par l’ARN polymémase: Affinités relatives du core enzyme et de l’holoenzyme pour l’ADN Initiation Holoenzyme Fixation au promoteur Elongation Core Abandon promoteur terminaison Core+fact term Décrochage de l’ADN Core + sigma Recyclage 36 Exemples de facteurs sigma chez E. coli 37 Initiation chez les eucaryotes 38 RNA Pol-I La synthèse du précurseur des ARN ribosomaux a lieu dans le nucléole 39 Initiation de la transcription par la RNA pol I (synthèse des ARN ribosomiques) Les ARN pol. eucaryotes ne se fixent jamais seules: facteurs de transcription Un « core factor » constitué de nombreux facteurs Core Factor TBP + pol I et Rrn3p Rrn3p Complexe d’initiation Pol I 40 ARN pol III: le gène 5S n’est pas contrôlé par un promoteur en amont Mise en évidence d’une région interne de contrôle de la transcription activité délétions + + + + + + ICR=Internal control region - - +1 +40 +80 +120 gène ARN 5S - - + + Mais comment la polymérase + Peut-elle se placer correctement? + délétions activité 41 Explication: le complexe d’initiation de la transcription de la pol-III TFIIIC TFIIIB Gène tRNA Pol III A B TFIIIC TFIIIB TFIIIA Gène ARNr 5S Pol III C 42 Séquence de la région ICR du gène codant pour l’ARN5S de Xenopus laevis et structure du facteur de transcription TFIIIA qui se fixe sur la région ICR Région ICR TFIIIA 12 acides aminés 43 Trois familles de doigts de zinc 44 Représentation schématique de l’interaction de l’ADN avec les doigts de Zinc en tandem contenus dans le facteur TFIIIA 45 La RNA pol. II 46 Détermination de la région promoteur d’un gène dépendant de l’ARN polymérase II Les régions sont délétées 47 Identification de 3 Régions nécessaires à l’initiation de la transcription, en amont du gène codant pour la β-Globine Expériences de mutations ponctuelles Boite Boite Boite GC CAAT TATA 48 Les promoteurs utilisés par la pol-II sont des combinaisons variées de courtes séquences consensus 49 Formation du complexe de démarrage pol-II Complexe préinitiation TATA + TBP + TFIIH Transcription basale Sans activation + TFIIB Pol II + TFIIE TFIIF + CTD 50 Eléments de contrôle du promoteur pol-II : les éléments distants enhancers promoteur gène enhancers UAS= upstream activating sequence 51 Comment fonctionne un enhancer? Modèle simplifié d’activation de la transcription Enhancer Activateur de transcription Boucle d’ADN TBP RNA Pol TATA box Site d’init. 52 TBP peut s’associer à des facteurs (les TAF) et former le complexe TFII-D: Activation de la transcription TAF250 TAF80 TAF110 TAF60 TAF30α TAF30β TAF40 TBP TAF150 TATA Initiateur TAF: TBP-Associated Factors : ouvrent la chromatine, acetylent histones etc. 53 Niveau basal / Niveau activé interactions TBP B F E H Transcription promoteur basale Activateurs TAF250 TAF80 TAF110 TAF60 TAF30αTAF30 β TAF40 TAF150 TBP B F E H Transcription activée promoteur 54 TBP est nécessaire à l’initiation de la transcription par chacune des 3 polymérases eucaryotes TBP TBP Pol III Pol II TBP Pol I 55 Structure de la TBP de levure en complexe avec une boite TATA Structure en « selle de cheval » Fixation dans sillon mineur 56 Un mélange d’interactions: - non-spécifiques (coller à l’ADN sur les groupements P) - spécifiques (reconnaître la séquence TATA) 57 Torsion de la double hélice d’ADN 58 Elongation de la transcription:
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